title: Transient kinetics of substrate binding to Na+/K(+)-ATPase measured by fluorescence quenching.
abstract: This paper examines the transient kinetics of substrate binding to the Na+/K(+)-ATPase labelled with iodoacetamidofluorescein (IAF) using fluorescence quenching by trinitrophenyl-ATP (TNP-ATP). Earlier work (E.H. Hellen, P.R. Pratap, 1996, Fluorescence quenching of IAF-Na+/K(+)-ATPase via energy transfer to TNP-labelled nucleotide, Proceedings of the VIIIth International Conference on the Na+/K(+)-ATPase, in press) has shown that TNP-nucleotide binds to specific sites (from which unlabelled nucleotide can displace it) and nonspecific sites (from which unlabelled nucleotide cannot displace it). Under stopped-flow conditions, quenching of IAF-enzyme fluorescence was well described by a stretched exponential (F(t) = F infinity + delta F exp[-Bt alpha]). Physically, this function may be interpreted in terms of its inverse Laplace transform phi (k), which describes a distribution of rate-constants; alpha reflects the width of this distribution. As TNP-ATP concentration increased, alpha decreased, reflecting TNP-ATP binding to sites with higher energy barriers. alpha decreased by about the same amount with increasing [TNP-ATP] in the presence of saturating ATP, indicating that the distribution of rate-constants is largely associated with the nonspecific binding sites. However, alpha was significantly less than 1 for ATP-induced fluorescence recovery in the presence of TNP-ATP, indicating that rate-constants associated with specific binding site are also distributed. The distribution of rate-constants for binding to the specific site indicates a distribution in the energy of the transition state for substrate binding. These results suggest that the specific binding site (in either the empty or the full state) may exist in a series of conformations separated by small energy barriers. However, the energy barriers for binding associated with these conformations are significantly distributed.
authors.affiliation: Department of Physics and Astronomy, University of North Carolina at Greensboro 27412, USA. pratapp@dirac.uncg.edu
authors.forename: P R
authors.initials: PR
authors.lastname: Pratap
authors.forename: E H
authors.initials: EH
authors.lastname: Hellen
authors.forename: A
authors.initials: A
authors.lastname: Palit
authors.forename: J D
authors.initials: JD
authors.lastname: Robinson
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004126:Dimethylformamide / Q000494:pharmacology; D004285:Dogs; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D005470:Fluorometry; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; C015296:5-iodoacetamidofluorescein; D004126:Dimethylformamide; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase